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The Journal of Cell Biology, Vol 131, 1831-1838, Copyright © 1995 by The Rockefeller University Press


ARTICLES

The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2

A Benmerah, J Gagnon, B Begue, B Megarbane, A Dautry-Varsat and N Cerf-Bensussan
Developpement Normal et Pathologique du Systeme Immunitaire, INSERM U 429; Hopital Necker-Enfants Malades, Paris, France.

The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2- terminal sequence and their immunoreactivity with anti-alpha- and anti- beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti- alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.
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